This project will investigate mechanisms that regulate sex specific transcription in Drosophila. This will be done by investigating the connection between the genetic, sex differentiation pathway and a target gene that is regulated by the pathway. An enhancer from a target gene directs female and tissue specific transcription in vivo. It is composed of three protein binding sites, one of which regulates sex specific transcription in a way that depends solely on the binding of the two doublesex gene proteins, DSX/F and DSX/M. The doublesex gene is at the bottom of the sex differentiation pathway. From this DNA site, DSX/F activates transcription in females and DSX/M represses transcription in males. This establishes the two doublesex proteins and their binding site in the enhancer as the connection between the pathway and a target gene. The broad aim of the project is to investigate the mechanisms by which DSX proteins regulate transcription from this enhancer. This will be done by examining the structure and function of DSX/F and DSX/M. First those functions of DSX proteins that can now be assayed by biochemical methods, by unicellular biological systems and by a few germline transformation experiments will be studied. In this work the dimerization and tetramerization domains will be localized and then amino acid changes that specifically block each oligomerization step will be identified. Hypotheses concerning the effect of DSX protein oligomerization on DNA binding and the effect of DNA binding on DSX oligomerization will be tested by physical methods using wild type proteins and proteins with mutants blocking oligomerization. Similar methods will be used to test hypotheses that the sex-specific termini of the proteins affect oligomerization and DNA binding. Germline transformation will be used to determine the regulatory functions of oligomerization and to localize the transcriptional activation domain of DSX/F. Genes for the other two regulatory proteins that operate from the enhancer will be isolated and used to develop in vitro and unicellular assays for DSX protein function. These assays will then be used to examine the structure and function of the DSX proteins.